1. Field of the Invention
This invention pertains to an apparatus and method for characterizing peptides and other biological moieties by electrospray ionization mass spectrometry of samples provided by high performance liquid chromatography (HPLC). A device is provided which permits broadening of HPLC elution peaks without loss of resolution, simultaneously slowing the solvent flow of the system without burdening the system with substantial dead volume for the HPLC eluent. The device also provides for application of the voltage necessary for sheathless electrospray ionization in a waste line thereby overcoming issues related to application of the voltage.
2. Background of the Prior Art
Mass spectrometry has rapidly developed as the method of choice for sequencing biologically derived molecules..sup.1,2 The high complexity of biological mixtures often makes coupling a separation technique, such as HPLC, highly desirable or even required..sup.2 Unfortunately, for many detection techniques including mass spectrometry, as the separation efficiencies increase, the peak widths tend to narrow placing more stringent speed requirements on the mass spectrometer (MS), making structural analysis of peptides by collisionally-activated dissociation (CAD) difficult.
Also important in many biological analysis is the detection limit of electrospray ionization MS techniques. Electrospray ionization (ESI) efficiencies with conventional ESI sources are compromised in two ways. First, as an analyte enters into the ionization region of the ESI source it is diluted by a sheath liquid to help stabilize the ESI process and apply the potential needed for ESI to the HPLC effluent..sup.3 Second, the large electrospray plume generated by the convention ESI source is sampled by a very small orifice leading into the MS giving low transmission efficiencies..sup.4 Recent advances in ESI techniques have shown improvements in ESI transmission efficiencies by eliminating sheath liquids and reducing total flows into the ESI source..sup.3-8
One of the most widely used miniaturized sheathless ESI sources (.mu.-ESI) is the Nanospray source of Mann et al..sup.5 This source uses glass capillaries pulled to &lt;5 .mu.m, sample flow rates at &lt;5-50 nL/min, and is capable of analyzing low femtomole amounts of sample in a volume of 1 .mu.L continuously for more than 1 hour. The long analysis time allows multiple MS experiments to be performed. This method, however, often requires substantial signal averaging (&gt;10 min) to acquire satisfactory S/N to identify precursor masses for subsequent CAD experiments and does not have the ability to interface with a separation technique. The tips for this source are fragile, expensive, non-reusable, and the position of the tip at the orifice into the mass spectrometer is critical, often requiring expensive camera equipment in order to achieve optimum signal.
The use of reverse phase HPLC on line with .mu.-ESI for the analysis of peptide mixtures offers numerous advantages over Nanospray ESI, including desalting and detergent removal, complicated mixtures can be time resolved, more dilute samples can be used because the sample is concentrated on column, and information on the hydrophobicity of the analyte can be obtained. However the short time window in which a peptide elutes can be problematic if multiple stages of MS are desired. In order to sequence biological peptides using MS, it is necessary to 1) identify the peptide and 2) dissociate the peptide such that it fragments randomly along the amide linkages. CAD on the ion trap MS involves several steps. The precursor ion must first be isolated using tailored rf waveforms applied to the endcap electrodes. The ion must then be activated by increasing the ions kinetic energy using rf waveforms applied to the endcaps in resonance with the ion in the, presence of a bath gas such that the ion collides with the bath gas converting kinetic energy into internal energy. The ion then may fragment and the fragments are scanned out and detected. This identification/CAD process can be 5 seconds while typical HPLC peak widths are ca. 12 seconds.
It has been shown that slowing the flow rate down during an HPLC run can satisfactorily increase the elution time of a peptide by as much as 10 times..sup.7,8 Potential problems with this method for commercial syringe pump HPLC systems are the time delay before the slow column flow is realized and the dead volume of the system after slow column flow is achieved. The time delay before slow column flow is achieved arises from the total volume of the system being at the running pressure of the column. To slow down the column flow the entire HPLC system pressure must be reduced to the desired low column flow pressure. The second potential problem is the dead volume after the solvent mixing tee. The desired HPLC gradient must pass through the dead volume after the mixing tee at the slow flow rates, this will give significant lag times in the HPLC gradient profiles. Davis et al. use a preformed gradient in a fused silica capillary (FSC) and a programmable ISCO syringe pump to slow down and speed up the flow rate of the mobile phase over the column..sup.7 To combat the lag time before slow flow is achieved they actually reverse the syringe pump to lower the column head pressure very quickly. The preformed gradient fixes the profile of the HPLC run before changing pressures such that they have no HPLC gradient lag times due to dead volumes. Unfortunately, these methods to overcome dead volume issues will not work with more common HPLC syringe pump systems.
Accordingly it remains an object of those of skill in the art to develop a process to characterize biological molecules and compounds by MS, particularly, through ESI MS. It is a further object of those of skill in the art to provide an apparatus that permits broadening of HPLC peaks in an ESI environment, without dead volume and at a slow or controlled flow rate.